The widespread exploitation of monoclonal antibodies for medical research activities, therapeutic treatments, and medical diagnostic immunoassays constitutes a multi-billion dollar health care industry. This project will investigate two types of novel synergistic binding reactions recently described for selected antibody- antigen pairs: those where the affinity of the antibody appeared to increase as the antigen concentration increased (positive cooperativity);and those where the affinity of an antibody directed toward one epitope on a protein antigen appeared to increase in the presence of a second antibody directed toward a different, separate epitope on the same protein antigen. Analytical experiments are proposed with the goal of understanding the molecular mechanisms of these novel synergistic binding reactions. Methods include, but are not limited to, analytical ultracentrifugation, asymmetric field flow fractionation, isothermal titration and differential scanning calorimetry, and circular dichroism and other spectroscopic measurements. Specific aims 1 and 3 are to conduct detailed structural and functional studies on monoclonal antibodies that exhibit synergistic binding behavior with their respective antigens. Practical goals include (i) the determination of antigen binding stoichiometries for antibodies that exhibit apparent Hill coefficients below and above 2.0 and (ii) the observation and correlation of antibody conformational changes to antigen binding. Specific aim 2 is to conduct functional and structural studies on protein antigens that bind monoclonal antibodies to separate epitopes in a synergistic manner. The principal goal of this aim is to quantify the relative contributions of both avidity effects and binding-dependent protein conformation changes to the apparent synergy of binding. In both aims, the unifying hypothesis is that the underlying molecular mechanisms are protein conformation changes that occur as a consequence of the antibody-antigen binding interaction. Any attempts to exploit antibodies that exhibit novel synergistic binding properties to enhance current clinical diagnostic or therapeutic applications must be dependent on existing knowledge concerning the molecular mechanisms whereby the novel binding is expressed. This project is expected to contribute fundamental knowledge toward manipulating these unexpected activities for diagnostic and therapeutic applications. It will also contribute to a basic understanding of a heretofore unrecognized aspect of antibody function.